H2-E transgenic class II-negative mice can distinguish self from nonself in susceptibility to heterologous thyroglobulins in autoimmune thyroiditis

 Susceptibility to experimental autoimmune thyroiditis (EAT) is linked to H2-A class II genes;k and s haplotypes are susceptible, while b and f are resistant. EAT is inducible with thyroglobulins (Tgs) from several mammalian species which share portions of identical sequences. But cross-activation and cross-tolerance studies with mouse (m), human (h), and porcine (p) Tg have indicated mTg-unique T-cell epitope(s), in addition to conserved, in EAT induction. The recent introduction of the HLA-DRB1*0301 (DR3) transgene rendered major histocompatibility complex (MHC) class II-negative (Ab⁰) mice susceptible to EAT induction by both hTg and mTg, suggesting usage of conserved epitopes. Here, we introduced the H2-Ea ^k transgene into resistant B10 (H2 ^b ) or Ab⁰ mice with a defective Ea gene to provide functional surface H2E (b haplotype) expression. Surprisingly, both transgenic strains showed severe inflammation only after hTg, but not mTg, immunization, although the moderating influence of the A ^b gene in B10 was evident. In proliferative assays, hTg-primed cells did not respond to mTg, nor to conserved 12mer peptides from three primary hormonogenic sites, two of which can activate T cells for thyroiditis transfer and cytotoxicity. The vigorous response to hTg stimulation was reduced only by Eβ^b-specific monoclonal antibody. EAT induction with bovine and pTg showed responses similar to hTg, suggesting thyroiditogenic epitopes shared with hTg, but not mTg. This is the first demonstration of: (1) nonpermissiveness for EAT induction with mTg, normally the most thyroiditogenic Tg and the one with unique epitopes for susceptible mice, and (2) the separation of hTg from mTg in EAT induction in H2-E-transgenic mice. Received: 15 January 1999 / Revised: 23 April 1999     

Mapping of thyroglobulin epitopes: Presentation of a 9mer pathogenic peptide by different mouse MHC class II isotypes

We have previously reported that the 17mer thyroglobulin (Tg) peptide TgP1 (a.a. 2495–2511) induces experimental autoimmune thyroiditis (EAT) inH-2 ^k mice, a process requiring expression ofE ^k genes, and inH-2 ^S mice that lack functional E molecules. To test whether this apparent discrepancy was due to recognition of distinct TgP1 determinants in each strain, we mapped in this study minimal T -cell epitopes within TgP1 and examined their pathogenicity in C3H (H-2 ^k) or SJL (H-2 ^S) mice. Truncation analysis using TgP1-specific, CD4+ hybridomas from C3H mice identified two overlapping determinants, (2496-2504) and (2499–2507), that were restricted by the E^k and A^k molecules, respectively. Subsequent challenge of C3H and SJL mice with these 9mer peptides revealed that the Ekrestricted (2496–2504) determinant elicited EAT and specific proliferative LNC responses in both strains, suggesting recognition in the context of A^s, since this is the only class II molecule expressed in SJL mice. This was further confirmed by blocking of the proliferative LNC response by an As-specific monoclonal antibody. In contrast, the A^k-restricted (2499–2507) determinant induced weak EAT and no proliferative LNC responses in either strain. These data 1) delineate the 9mer (2496–2504) peptide as a minimal Tg T-cell epitope with direct pathogenic potential in mice and 2) highlight the use of nonisotypic MHC class II molecules for the presentation of this peptide in mice of differentH-2 haplotypes.     

The role of MHC class I heterodimer expression in mouse ankylosing enthesopathy

 Ankylosing enthesopathy (ANKENT) is a spontaneous mouse joint disease with strikingly similar pathology to human HLA-B27-associated enthesopathies such as ankylosing spondylitis. In C57Bl/10 mice, transgenic HLA-B^*2702 as well as H2 genes have been shown to be relative risk factors for ANKENT. To investigate the role of major histocompatibility complex (MHC) class I expression in disease pathogenesis, ANKENT occurrence was compared among β₂-microglobulin (β₂m) knockout littermates with or without transgenes for HLA-B^*2702 and human β₂m. In the knockout phenotype lacking β₂m, ANKENT occurrence is significantly reduced (P = 0.016). In the absence of β₂m, B^*2702 is not detected on the cell membrane, nor does it increase the risk for ANKENT. This means that the previous finding that HLA-B^*2702 increases susceptibility to ANKENT in C57Bl/10 mice cannot be ascribed to a transgene insertion effect. Rather, in order to increase disease susceptibility, B^*2702 must be coexpressed with mouse β₂m (mo-β₂m). In contrast, when HLA-B^*2702 is expressed with β₂m of human origin, disease susceptibility is not affected. Thus, both H2^b-derived class I heterodimers and HLA-B^*2702/mo-β₂m heterodimers contribute to ANKENT susceptibility.     

Distinct genetic pattern of mouse susceptibility to thyroiditis induced by a novel thyroglobulin peptide

Experimental autoimmune thyroiditis (EAT), induced by thuroglobulin (Tg) and adjuvant, is major histocompatibility complex-controlled and dependent on Tg-reactive T cells, but the immunopathogenic T-cell epitopes on Tg remain mostly undefined. We report here the thyroiditogenicity of a novel rat Tg peptide (TgP2; corresponding to human Tg amino acids 2695–2713), identified by algorithms as a site of putative T-cell epitope(s). TgP2 causes EAT in SJL (H-2 ^s) but not in C3H or B10.BR (H-2 ^k), BALB/c (H-2 ^d), and B10 (H-2 ^b) mice. This reveals a new genetic pattern of EAT susceptibility, since H-2 ^k mice are known to be high reponders (susceptible) after Tg challenge. Following in vivo priming with TgP2, T cells from only SJL mice proliferated significantly and consistently to TgP2 in vitro, whereas TgP2-specific IgG was observed in all strains tested. Adoptive transfer of TgP2-primed SJL lymph node cells to naive syngeneic recipients induced a pronounced mononuclear infiltration of the thyroid, which was more extensive than that observed after direct peptide challenge. TgP2 is non-immunodominant, since priming of SJL mice with rTg did not consistently elicit T-cell responses to TgP2 in vitro and a TgP2-specific T-cell hybridoma did not respond to antigen presenting cells pulsed with rTg. The data support the notion that Tg epitopes need not be either iodinated or immunodominant in order to cause severe thyroiditis and that the genetic pattern of the disease they induce can be distinct from that of Tg-mediated EAT. Correspondence to: G. Carayanniotis.     

Characterization of a novel H2A(-)E+ transgenic model susceptible to heterologous but not self thyroglobulin in autoimmune thyroiditis: thyroiditis transfer with Vbeta8+ T cells.

Recently we reported on a novel H2E transgenic, IA-negative model of experimental autoimmune thyroiditis (EAT) that excludes reactivity to self in its susceptibility pattern to heterologous thyroglobulin (Tg). In conventional, susceptible mouse strains, EAT is inducible with both homologous and heterologous Tg; e.g., human (h)Tg shares conserved thyroiditogenic epitopes with mouse (m)Tg. However, when an H2Ea(k) transgene is introduced into class II-negative B10.Ab(0) mice, which express neither surface IA (mutant Abeta-chain) nor surface IE (nonfunctional Ea gene), the resultant H2E(b) molecules are permissive for EAT induction by hTg, but not self mTg. Also, the hTg-primed cells do not cross-react with mTg. To explore this unique capacity of E+B10.Ab(0) mice to distinguish self from nonself Tg, we have developed T cell lines to examine the T cell receptor repertoire and observed a consistent Vbeta8+ component after repeated hTg stimulation. Enrichment and activation of Vbeta8+ T cells by either superantigen staphylococcal entertoxin B or anti-Vbeta8 in vitro enabled thyroiditis transfer to untreated A-E+ recipients, similar to hTg activation. Vbeta8+ T cells isolated by FACS from hTg-immunized mice also proliferated to hTg in vitro. These studies support the contribution of Vbeta8 genes to the pathogenicity of hTg in this H2A-E+ transgenic model.     

Structural and genetic properties of the Eb recombinational hotspot in the mouse

The Eb gene of the mouse contains a recombinational hotspot which plays a predominant role in meiotic crossing-over within the I region of the mouse major histocompatibility complex (MHC). The nucleotide sequences of five recombinants derived from H-2 ^k /H-2 ^b heterozygotes at the Eb locus placed the sites of recombination in each recombinant haplotype within a 2.9 kilobase (kb) segment located fully within the second intron of the Eb gene. Further resolution of the crossover sites was not possible since the nucleotide sequences of the parental and recombinant haplotypes are identical within this segment. The molecular characterization of these five recombinants considered in conjunction with three previously reported intra-Eb recombinants indicates that there are at least two distinct sites of recombination within the Eb recombinational hotspot. In a related study, an examination of the nucleotide sequence of the H-2 ^p allele of the Eb gene revealed a major genetic rearrangement in the 5' half of the intron in this haplotype. A 597 base pair (bp) nucleotide sequence found in the H-2 ^p haplotype is replaced by a 1634 bp segment found in the H-2 ^b and H-2 ^k haplotypes. Sequence analysis of this 1634 bp segment shows strong nucleotide sequence similarity to retroposon long terminal repeat (LTR), env, and pol genes indicating that this segment of the second intron has evolved through retroposon insertion. The location of these retroposon sequences within the 2.9 kb recombination segment defined by the five H-2 ^k /H-2 ^b recombinant haplotypes suggests a possible relationship between these retroviral elements and site-specific recombination within the second intron of the Eb gene. Offprint requests to: H. C. Passmore     

Polymorphism of the MHC class II Eb gene determines the protection against collagen-induced arthritis

Collagen-induced arthritis (CIA) is an animal model of auto immune polyarthritis, sharing similarities with rheumatoid arthritis (RA). Paradoxally, susceptibility to mouse CIA is controlled by the H2A loci (DQ homologous) while RA is linked to HLA.DR genes (H2E homologous). We recently showed that the E^d molecule prevents CIA development in susceptible H2 ^q mice. We addressed the question of whether H2Eb polymorphism will influence CIA incidence as HLA.DRB1 polymorphism does in RA. In F₁ mice, only H2Eb^d and H2Eb^s molecules showed protection. Using recombinant B10.RDD (Eb ^d/b) mice, we found that CIA protection was mediated by the first domain of the E^d molecule. Using peptides covering the third hypervariable region of the E chain, we found a perfect correlation between presentation of E peptides by the H2A^q molecule and protection on CIA. Therefore, the mechanism by which H2Eb protects against CIA seems to rely on the affinity of E peptides for the H2A_q molecule.     

Histocompatibility gene mutation rates in the mouse: a 25-year review

 Mutation rates of H2 and non-H2 histocompatibility genes in the mouse are examined over a 25-year period. Detected by skin graft rejections, the mutations were screened in inbred and hybrid mice from a continuously maintained and monitored colony and from a regularly supplied set of mice provided from the National Cancer Institute for monitoring of genetic integrity. Twenty-five H2 mutations were recovered, involving the K, D, L, and Ab loci, as well as over 80 mutations of non-H2 histocompatibility genes. Aside from a single allele at a single locus (H2-K ^b ), the spontaneous mutation rate of H2 class I genes appears to be equivalent to that found estimated for non-H2 histocompatibility genes, and comparable to rates reported for a variety of mouse genes. This is in contrast with previous suggestions that H2 genes mutate at orders of magnitude greater than do “average” mammalian genes. The discrepancy is attributed to the H2-K ^b gene which accounts for over half of all reported H2 mutations and which mutates spontaneously at a rate of 1–2×10^–4 per gene per generation. Furthermore, over half of the spontaneous H2-K ^b mutations result in a single mutant phenotype (the “bg” group) which involve similar changes at amino acid residues 116 and 121. Thus, the high spontaneous mutation rate for H2-K ^b appears to be the exception among major histocompatibility genes, rather than the rule. Received: 18 April 1997 / Revised: 22 May 1997     

Both α and β chain polymorphisms determine the specificity of the disease-associated HLA-DQ2 molecules,with β chain residues being most influential

 We compared the peptide binding specificity of three HLA-DQ molecules; HLA-DQ(α1^*0501, β1^*0201), HLA-DQ(α1^*0201, β1^*0202), and HLA-DQ(α1^*0501, β1^*0301). The first of these molecules confers susceptibility to celiac disease and insulin-dependent diabetes mellitus, while the two latter molecules, which share either the α chain or the nearly identical β chain with HLA-DQ(α1^*0501, β1^*0201), do not predispose to these disorders. The binding of peptides was detected in biochemical binding assays as inhibition of binding of radiolabeled indicator peptides to affinity-purified HLA-DQ molecules. Binding experiments with several peptides demonstrated a clear difference in peptide binding specificity between the three HLA-DQ molecules. Further, single amino acid substitution analyses indicated that the HLA-DQ molecules have different peptide binding motifs. The experimental data were corroborated by computer modelling analysis. Our data suggest that the three HLA-DQ molecules prefer large hydrophobic residues in P1 of peptides with subtle differences in side-chain preferences. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202) both prefer large hydrophobic residues in P9, whereas HLA-DQ(α1^*0501, β1^*0301) prefers much smaller residues in this position. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202), in contrast to HLA-DQ(α1^*0501, β1^*0301), prefer negatively charged residues in P4 and P7. A less prominent P6 pocket also appears to differ between the three HLA-DQ molecules. Our results indicate that polymorphic residues of both the α and the β chain determine the peptide binding specificity of HLA-DQ(α1^*0501, β1^*0201), but that the β chain polymorphisms appears to play the most important role. The information on peptide residues which are advantageous and deleterious for binding to these HLA-DQ molecules may make possible the prediction of characteristic features of peptide that bind to HLA-DQ(α1^*0501, β1^*0201) and precipitate celiac disease. Received: 2 July 1996 / Revised: 7 August 1995     

Possible role of Ab b gene in mouse resistance to EL4 metastases

S (survivor) mutants were produced in mice for genetic analysis of host resistance to metastatic cancer. S-mutants S-27 and S-31 resist transplantation of lymphoma EL4 of parental C57BL/6J (B6) mice while they accept parental skin grafts. Mutant S-27 also resists formation of spontaneous metastases from intradermally growing EL4 tumor into lymp nodes; mutant S-31 is highly susceptible to EL4 metastases. Another mutant. H-2 ^bm26 (bm26), resists EL4 and rejects B6 skin grafts. Major histocompatibility complex (MHC) class I and class II gene expression was compared in these mutants and normal B6 mice. All three mutants tested, S-27, S-31, and bm26, expressed a low amount of K ^b mRNA in organspecific fashion. Mutant bm26 and S-31 expressed a low amount of Ab ^b mRNA and of A^b antigen on their spleen cells. Some oligonucleotide probes designed to hybridize to the second exon of the clss II MHC gene Ab ^b did not hybridize with DNA from all three mutants. These findings suggest extensive sequence alternations in the Ab ^b gene in mutants S-27, S-31, and bm26; they also suggest a major role of MHC in the control of host resistance to spontaneous metastases of the EL4 tumor. Address correspondence and offprint request to: I. K.. Egorov.     

Intrachromosomal gene conversion frequency in the H2 differs between haplotypes

 We examined two intrachromosomal gene conversion events with a polymerase chain reaction assay at the DNA level between the two major histocompatibility complex class II genes Eb and Ab in mice sperm before selection has occurred. The frequency of the intrachromosomal gene conversion event between Ebd and Abd was found to be at least one order of magnitude higher than between Ebk and Abk in the same mice. Parental imprinting of the genes appears not to have an effect on gene conversion, as both (d×k)F₁ and (k×d)F₁ mice have indistinguishable frequencies in both haplotypes. The number of DNA copies of the donor and acceptor genes present in the cell at the time of mutation does not seem to influence the frequency of the intrachromosomal gene conversion in the k haplotype, whereas the frequency in the d haplotype is increased when double the number of donor and acceptor genes is present. The DNA fragment transferred between Ebd and Abd is invariably short, and need not comprise more than six nucleotides. The fragment transferred within the k haplotype varies in length, and can attain at least 100 nucleotides. The difference between the haplotypes both in length and frequency might be attributed to a six-nucleotide deletion in the Abk gene, which might make base-pairing between the genes less efficient and less precise. Received: 24 October 1997 / Revised: 5 January 1998     

A novel MHC class I-related molecule expressed on mouse thymic stroma cells and mature lymphocytes

A monoclonal antibody (mAb) TP-3 has been established by immunizing rats with the BALB/c mouse thymic epithelial cell line TEL-2. The TP-3 antigen is expressed on stroma cells of thymus, spleen, and lymph node in syngeneic BALB/c mice (H-2 ^d ). This antigen is also expressed at a low level on the cell surface of immature thymocytes, and at a high level on mature T and B cells. In allogeneic mice such as C57BL/6 (H-2 ^b ) or C3H (H-2 ^k ), no cells expressed the TP-3 antigen. Using H-2 congenic mice, reactivity with mAb TP-3 was found to map to a region of H-2D ^d L ^d or between D ^d and Qa, suggesting that TP-3 is a major histocompatibility complex (MHC) class I antigen. However, immunoprecipitation analysis indicated that this antigen is not identical to the classical mouse class I molecules in terms of molecular size, antigenicity, and tissue distribution.     

Levels of Endogenous lnterleukin-1, Interleukin-6, and Tumor Necrosis Factor in Congenic Mice Infected with Borrelia garinii

This study describes the levels of interleukin-1 alpha (IL-1α), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) in the sera and parenchymal organs of various congenic mouse strains infected with Borrelia garinii. A significant elevation of inflammatory cytokine levels was found in the organs of C3H/HeN (H-2^k) and B10.BR (H-2^k) mice but not in those of BALB/c mice (H-2^d). Focally produced cytokines can contribute to antimicrobial defense against these organisms. High levels of IL-1α were observed in the sera of C3H/HeN, B10.BR and B10 (H-2^b) mice infected with B. garinii and they were associated with the presence of spirochetes in the skin. Thus, susceptible mice demonstrated a stronger cytokine response than resistant mice. This study presents in vivo evidence that B. garinii infection affects the immunopathogenesis of Lyme disease.     

H-2 class I antigen expression on mouse teratocarcinoma cell lines

Immunity against PCC3 teratocarcinoma cells (129, H-2 ^b) was induced in allogeneic (C3H, H-2 ^k) mice by preimmunization with L cells (C3H, H-2 ^k) expressing cosmid-introduced K ^b or D ^b genes, but not with nontransfected L cells. In addition, the growth of PCC3 cells in sublethally irradiated (C3H × B6-H-2 ^bm1)F₁ and (C3H × B6-H-2 ^bm13 )F₁ mice bearing the K ^bm1 and D ^bm13 mutations, respectively, was either prevented, stopped, or delayed in comparison with the (C3H × B6)F₁ (k × b) mice, which failed to reject the PCC3 cells. The teratocarcinoma line OC15S was exceptional because it reacted specifically with K^b- and D^b-specific (but not I^b-specific) alloantisera, and because K^b- and D^b-specific antibodies could be absorbed by OC15S cells. The subpopulation of OC15S cells bearing the ECMA-7 antigen characteristic for embryonic carcinoma (EC) cells was isolated by the fluorescence-activated cell sorter and was shown to react specifically with K^b- and D^b-specific antisera. These experiments show that teratocarcinoma cells express antigens similar or identical to the K-and D-region products of differentiated cells. The lack of expression of class I antigens is thus neither a condition nor a consequence of the pluripotentiality of the EC cells. The exact nature of the major histocompatibility complex antigens on EC cells has yet to be established using the methods of molecular biology and biochemistry.     

Intrahaplotype and interhaplotype pairing of bovine leukocyte antigen DQA and DQB molecules generate functional DQ molecules important for priming CD4+ T-lymphocyte responses

Antigen-specific CD4⁺ T-lymphocyte responses are restricted by major histocompatibility complex class II molecules, which influence T-cell priming during infection. Human leukocyte antigen (HLA) and bovine leukocyte antigen (BoLA) DRB3 and DQ genes are polymorphic, but unlike HLA, many BoLA haplotypes have duplicated DQ genes, and antibody-blocking studies indicated that BoLA-DQ molecules present various pathogen epitopes. Limited experimentation also suggested that BoLA-DQ molecules formed by interhaplotype pairing of A and B chains are functional. To compare antigen presentation by DR and DQ molecules and to definitively demonstrate functional BoLA-DQ molecules derived from interhaplotype pairing, different combinations of DR or DQ A and B proteins were expressed with CD80 in 293-F cells for use as antigen-presenting cells (APCs). This approach identified 11 unique restriction elements including five DR and six DQ pairs for antigen-specific CD4⁺ T-cell responses against tick-transmitted bovine hemoparasites Anaplasma marginale or Babesia bovis. Interhaplotype pairing of DQ A and B molecules was demonstrated. Testing of six expressed DQA/B pairs from an animal with duplicated DQ haplotypes (DH16A/DH22H) demonstrated that an interhaplotype pair, DQA*2206/DQB*1301, presented A. marginale peptide B. In DH22H and DH16A homozygous animals, DQA*2206 was tightly linked with DQB*1402, and DQA*22021 was linked with DQB*1301. APCs from these donors could not present peptide B, confirming that DQA*2206/DQB*1301 encoded a functional interhaplotype pair. Functional BoLA-DQ molecules are generated by both intrahaplotype and interhaplotype pairing of A and B chains and play a similar role to BoLA-DR in priming helper T-cell responses to important pathogens.     

Variable spread of X inactivation affecting the expression of different epitopes of the Hya gene product in mouse B-cell clones

Cloned B-cell lines from a female T16H/XSxr mouse in which Tdy expression was suppressed due to X inactivation and from a male X/XSxr mouse, both of the (kxb)F₁ haplotype, were examined for H-Y expression. This was determined both by their ability to act as targets for H-2^k and H-2^b-restricted H-Y-specific cytotoxic T cells and by their ability to stimulate the proliferation of H-2K^k, H-2D^b (class I) and A^b (class II)-restricted T-cell clones. In B-cell clones from the T16H/XSxr mouse, expression of H-Y/D^b exhibited partial X inactivation and only a proportion ( 30%) of the cells were targets for or stimulated H-2D^b-restricted H-Y-specific T cells. In contrast, H-Y eiptopes restricted by H-2^k (H-Y/K^k, H-Y/D^k) and A^b (H-Y/A^b) exhibited no X inactivation. Furthermore, no inactivation of H-Y/D^b, H-Y/A^b, or H-Y^k was observed in the male X/XSxr mouse. These results indicate that the T16H/XSxr female is a mosaic, as a result of the variable spread of X inactivation into the Sxr region. They further suggest that the H-Y antigen recognized in association with H-2^k and H-2D^b class I molecules and A^b class II molecules may be the product of more than one gene.     

MHC class II Ab diabetogenic residue 57 Asp/non-Asp dimorphism influences T-cell recognition and selection

 Human and mouse major histocompatibility complex class II beta chain alleles associated with predisposition to type I diabetes often encode a non-charged residue at position 57 rather than the negatively charged aspartate residue characteristic of non-susceptible haplotypes. The mechanism(s) whereby this polymorphism promotes eventual pancreatic beta cell destruction is unclear. The type I diabetes-susceptible mouse strain NOD (H2^g7) encodes serine at Ab position 57 and is one of the few mouse class II molecules not encoding aspartate at this position. To gain insight into the structural impact of this amino acid substitution and any influence it may have on T-cell selection, we assessed whether T-cell repertoires selected by diabetogenic class II (A^g7) are tolerant of mutant Ab (residues 56 and 57) H2-A^g7. We find that NOD mice mount an allogeneic response to skin grafts expressing mutant position 57 (serine to aspartate) Ab^g7; but not to grafts expressing mutant position 56 (histidine to proline) Ab^g7. Graft rejection correlates with the presence of CD4⁺ T cells specific for the mutant H2-A^g7 heterodimer. Genetic analyses are consistent with Ab position 57 aspartate/non-aspartate dimorphism influencing peptide selection and hence repertoire selection. Direct evidence for the serine to aspartate substitution at position 57 influencing T-cell selection is found by analysis of peripheral T-cell receptor (TCR) usage and the CD4/CD8 T-cell ratio. Received: 18 June 1997 / Revised: 18 September 1997     

H2-A polymorphism contributes to H2-Eβ-mediated protection in collagen-induced arthritis

 Collagen-induced arthritis (CIA) is an animal model of auto-immune inflammatory polyarthritis which has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC) and is restricted to the H2 haplotypes q and r. In previous experiments, we have found that the introduction of an H2-Eb ^d transgene in H2-A^q CIA-susceptible mice was able to protect these mice against disease development. More recently, we have proposed that the polymorphism of the first domain of the Eβ molecule modulates this protection, and that the presentation of a peptide from the third hypervariable region of the Eβ chain by the H2-A^q molecule plays an important role in this mechanism. In the present report, we investigated whether the H2-E-mediated protection is H2-A^q-specific and whether the source of collagen has any influence. While the source of collagen had no effect on the protection, our results showed that the H2-E molecule failed to protect B10.RIII (H2^r) mice against CIA. Further, the H2 haplotype r exerted a negative effect on the Eβ^d-mediated protection in H2-A^q-restricted disease. Our results provide additional proof that self-MHC-derived peptides, such as Eβ peptides, may play an important role in the T-cell repertoire education and/or modulation of the T-cell response in the periphery. Received: 29 May 1996 / Revised: 26 June 1996     

TrackingH-2 alleles in transgenic mice by RFLP and heteroduplex analysis

Transgenic mice provide valuable tools for biological research including many areas of immunology. In studies involving the major histocompatibility complex (MHC), it is often necessary to place the desired transgene in a specificH-2 (the murine MHC) environment. In this regard, the strains commonly used for the production of transgenic mice also carry well characterizedH-2 alleles and provide an appropriate genetic background for MHC related experiments. In this study, a highly polymorphic microsatellite of tetranucleotide repeats from the second intron of the class IIEb gene within theH-2 complex was used in order to identify the corresponding alleles. The relevantH-2 allele(s) along with the transgene were then tracked throughout the production of a chicken ovalbumin-specific transgenic strain. The technique involved PCR-amplification of a DNA sequence encompassing theH-2 specific microsatellite followed by RFLP and heteroduplex analyses. This approach is likely to find wide application in the background checking of trangenic mice, especially in immunological research requiring a definedH-2 background.